The differences in glucose assimilation under light and dark conditions
The A. protothecoides was cultured in a 100 mL shake flask using conventional nitrogen-limited heterotrophic medium. The experimental group was placed under light (mixotrophic culture - MC) and the control group was placed in the dark (heterotrophic culture - HC). The biomass and residual glucose concentration in the medium were measured to compare the growth and glucose consumption of the cultures. As shown in Fig. 1A, the growth of the MC was slower than that of the HC, which depleted glucose on the 5th day. By contrast, glucose was not exhausted on the 7th day in the MC (Fig. 1B). The residual glucose concentration in the MC was higher than in the HC, indicating that A. protothecoides assimilated less glucose in the light. Many microalgae, such as Phaeodactylum tricornutum and Chlorella vulgaris, can use glucose when grown in light [22, 23]. When such organisms are grown in illuminated mixotrophic culture, the biomass will be higher due to the concomitant activity of both photosynthesis and organic carbon assimilation. Therefore, the phenotype of A. protothecoides of reduced growth and glucose assimilation in the light is different from other microalgae. The specific cell growth rates of A. protothecoides cells in autotrophic and heterotrophic mode were found to be 0.0028 and 0.0257 h-1, respectively . The specific cell growth rate in heterotrophic mode was 9 times higher than in autotrophic mode, indicating that carbon fixation via photosynthesis contributed less to the increase of biomass when both light and glucose were present.
Nitrogen is an essential element for chlorophyll and protein synthesis. Consequently, a sufficient supply of nitrogen sources can promote photosynthesis in microalgae . After increasing the concentration of the nitrogen source to a high level (e.g. 5 g/L glycine), the growth rate and glucose consumption were also reduced under illumination (Fig. 1C, D). The chlorophyll content was highest in the light with 5 g/L glycine, while the cells contained almost no chlorophyll in the dark with 0.5 g/L glycine (Fig. 1E). Furthermore, cells grown in the dark with 5g/L glycine and in the light with 0.5 g/L glycine had low levels of chlorophyll. The chlorophyll content under high nitrogen source concentrations was significantly higher than under low nitrogen, but the growth rate and glucose consumption observed at the high nitrogen source concentration was also inhibited by light. Therefore, the reduced accumulation of biomass and inhibition of glucose in the light was not simply caused by the photosynthetic fixation of carbon dioxide.
A. protothecoides can efficiently import glucose from the medium via a specific glucose transporter system. Gao et al. identified nine transporter homologs in A. protothecoides using the HUPs (H+/hexose co-transporter) from Chlorella kessleri as the query sequence . Moreover, three of the identified HUP-like proteins might be responsible for the ability of A. protothecoides to rapidly import glucose. Radiolabelled substrate can be used to measure the rate of sugar transport and the ability of sugar uptake more accurately. The sucrose transporter ST SUT1 from potato (Solanum tuberosum) was expressed in yeast and the transport activity and post-translational modification of ST SUT1 were measured using 14C-Sucrose . Unlike the tissues of higher plants, the unicellular A. protothecoides can be directly used to measure the glucose transport rate in vivo. A. protothecoides Al64 is an autotrophy-deficient strain obtained by ethyl methanesulfonate mutagenesis. The glucose transport activity of wild-type A. protothecoides and Al64 was measured using radiolabelled D-[2-3H] glucose. In Fig. 1F, the Y coordinate represents the radioactivity generated by D-[2-3H] glucose, which had been transported into cells within 3 minutes. This value reflects the glucose transport rate. The results showed that the glucose transport rate of wild-type cells in the dark was almost two times higher than under illumination (P=0.013 < 0.05), indicating that light could inhibit the glucose transport. By contrast, there was no significant difference in Al64 under the light and dark conditions. Hence, the glucose transport activity of Al64 was not affected by light, indicating that the inhibition of the glucose transport activity of A. protothecoides by light is related to the photosynthesis system and may be adjusted by changes in the intracellular environment.
The lipid content of A. protothecoides cells grown in heterotrophic mode four times higher (55.20%) than in the autotrophic mode . The heterotrophically grown cells were filled with large lipid droplets, while the lipid droplets in the autotrophically grown cells were smaller and fewer . Subsequently, the differences of subcellular structure between cells from the MC and the HC were observed by TEM (see Additional file 1). The structure of chloroplast was not found in cells from the HC, while incomplete chloroplast’ structure containing was observed in cells from the MC, indicating that structure of chloroplast was still retained under nitrogen limitation condition combined with illumination. The diameter of lipid droplet was larger than 2 μm in cells from the HC and smaller than 2 μm in those from the MC. The differences of starch, lipid and protein contents between cells from the HC and the MC were measured (Fig. 2). The starch and lipid contents of the MC were lower than that of the HC, indicating that lipid accumulation was inhibited under illumination (Fig. 2A, B). The protein contents of the MC were higher than that of the HC (Fig. 2C). This may be due to photosynthesis system need more proteins in the MC than HC. Under nitrogen limitation, most of the precursors for lipid biosynthesis were derived from extracellular glucose. The difference of lipid and protein accumulation between the HC and MC indicated that light also had an influence on glucose metabolism (see Additional file 1).
Comparative proteomic analysis of A. protothecoides cells grown under illumination and in the dark
Comparative proteomics was used to study the mechanism by which light regulates glucose uptake in Auxenochlorella protothecoides. Protein samples were labeled with TMT, identified by tandem mass spectrometry, and analyzed using the A. protothecoides protein database. A total of 4,820 proteins were identified in the samples, accounting for 68.5% of the total known proteins of A. protothecoides. The proteins with ratios greater than 1.3 or lower than 0.77 and p-values of less than 0.05 were identified as the differentially expressed proteins (DEPs) between the HC and MC. A total of 240 up- and 98 downregulated proteins were identified (see Additional file 2).
The online functional annotation tool DAVID was used to analyze the up- and downregulated proteins separately . The downregulated proteins were grouped into 3 clusters (Fig. 3A). The proteins in Cluster1 (Enrichment Score: 3.45) were mainly related to the biosynthesis and metabolism of lipids and fatty acids, which was consistent with the observed smaller lipid droplets in the cells from the MC. The proteins in Cluster2 (Enrichment Score: 1.67) were identified as having a non-covalent interaction with flavin adenine dinucleotide (FAD), which is also involved in the metabolism of pyruvic acid, fatty acids, oxidative degradation of amino acids and the electron transport chain. Cluster3 (Enrichment Score: 0.004) included integral membrane proteins and transmembrane proteins. The upregulated proteins were grouped into 8 clusters (Fig. 3B). Cluster1 (Enrichment Score: 4.58) contained many ribosomal proteins involved in the protein translation process. The upregulation of ribosomal proteins might be related to the increased requirements for proteins due to the construction of photosynthesis systems in the MC. The proteins of Cluster2 (Enrichment Score: 2.98) were associated with the photosynthesis system. It was shown that even under nitrogen-limited conditions (glycine concentration = 0.5 g/L), light could still promote the construction of the photosynthesis system. Cluster3 (Enrichment Score: 1.26) included iron-sulfur proteins and metal-binding proteins. Most proteins in this cluster were related to the electron transport chain of photosynthesis, indicating that the photosynthetic electron transport chain became active. Cluster4 (Enrichment Score: 1.2) was also related to photosynthesis. Importantly, all of the four clusters were consistent with the observed phenotypes, indicating that A. protothecoides needs to synthesize more proteins in MC to construct the photosynthesis system. Cluster 5 (Enrichment Score: 0.62) included proteins with sulfide oxidoreductase activity and those involved in intracellular redox balance. Thiol-disulfide exchange reduction/oxidation (redox) reactions regulate carbon fixation, respiration, and metabolic processes in plant cells . The redox system plays a key role in maintaining cellular homeostasis and regulating cell growth and metabolism . Under illumination, the redox homeostasis-related proteins in A. protothecoides were upregulated, indicating a change in intracellular redox balance. Hence, the redox balance was changed by light, and the change of cell homeostasis provided new clues for studying the influence of light on glucose assimilation. The enrichment scores of clusters 6, 7, and 8 were respectively only 0.37, 0.35 and 0.15, indicating that the enrichment of protein functions in these three clusters was poor.
Since A. protothecoides and Chlamydomonas reinhardtii are closely related, the protein-protein interaction (PPI) database of C. reinhardtii from STRING was used to analyze the interaction relationships of DEPs of A. protothecoides based on homology alignment . The DEPs in the PPI network numbered in accordance with the accession numbers of the proteins in the UniProt database (Fig. 3C). The different colors indicate that they were clustered according to similar functions or compositions. The red cluster contained to ribosome-associated proteins. The purple cluster was involved in carbon metabolism, indicating that light had an effect on carbon metabolism. Most proteins in the blue cluster were oxidoreductases. Proteins in the green cluster were enzymes associated with the tricarboxylic acid cycle. The purple-red cluster contained proteins involved in the synthesis of lipids. A yellow cluster was associated with fatty acid metabolism. Proteins in the orange cluster were associated with chlorophyll synthesis. Importantly, the results of PPI network analysis were consistent with the results of DAVID analysis.
The DEPs indicated by gray circles were not clustered. The largest gray circle was A0A087SDG3, which had the most interactions with other ungrouped proteins. A0A087SDG3 was predicted to be a thioredoxin reductase, and it was upregulated. Thioredoxin reductase, thioredoxin and NADPH constitute the thioredoxin system, which plays an important role in maintaining the intracellular redox balance. Although thioredoxin reductase was not clustered in the PPI network, the number of interacting proteins reflects a wide range of effects on DEPs. Combined with the analysis of the upregulated proteins in Cluster5, the DEPs and PPI information all reflected the significant changes in the expression of proteins related to the redox balance under illumination. This suggested that the intracellular redox balance was affected by light and this effect was likely related to the observed regulation of glucose assimilation.
Pathway mapping of DEPs in KEGG
The DEPs were uploaded to KEGG Mapper for pathway enrichment, whereby the upregulated proteins were marked in red and the downregulated proteins were marked in blue . The annotated proteins in the A. protothecoides genome were marked in green. According to the phenotype of influencing glucose assimilation under illumination, KEGG pathway analysis focused on photosynthesis, glycolysis, fatty acid biosynthesis and carbon fixation.
There were obvious differences in the subcellular structure of cells from the MC and the HC. Chloroplastic structures were apparent in the cells form the MC. While these were not as distinct as in the autotrophically grown cells, they nevertheless indicated that there was active photosynthesis in the MC (Fig. 2). All proteins enriched in the photosynthesis pathway were upregulated, including subunits of photosystem I (PSI), photosystem II (PSII) and the cytochrome b6-f complex (see Additional file 1). All top forty proteins were almost related to photosynthesis, indicating that light could promote the construction of the photosynthetic systems even under nitrogen-limited culture conditions. In addition to the constituent proteins of the photosystem reaction center, there were three oxygen-evolving enhancer proteins (OEE) among the top eight upregulated proteins. The upregulation fold changes of PsbO (OEE1), PsbP (OEE2) and PsbQ (OEE3) reached 8.09, 6.96, and 6.21, respectively. OEE is an important component of the oxygen-evolving complex (OEC) in PSII, which together with manganese clusters participates in the water splitting reaction to generate oxygen and electrons. Under NaCl- and heat stress, the extended structure of OEE can protect the reaction center D1 protein from oxygen radicals [32, 33]. Forster et al. found that two very high light (VHL)-resistant mutants of C. reinhardtii upregulated OEE1 expression under high light stress . OEE was also found to have a thioredoxin-like activity in Chlorella . Three OEE proteins were highly upregulated in MC, which could improve the function of intracellular OEC to maintain the activity of PSII and redox balance. Under nitrogen-limited conditions, the coexistence of glucose and light may be a stress factor for A. protothecoides cells. In response to stress, intracellular metabolism undergoes a series of adjustments that alter the accumulation of metabolites.
Light has an influence on glucose assimilation, including glucose transport and metabolism. Exogenous glucose first enters the glycolysis pathway to produce pyruvate (Fig. 4). Most of the DEPs in the glycolysis pathway were downregulated. Two enzymes that catalyze the production of D-glyceraldehyde 3-phosphate from D-glucose 6-phosphate, as well as pyruvate kinase were downregulated, which may eventually reduce the synthesis of pyruvate. The enzymes that are responsible for the producing acetyl-CoA from pyruvate, i.e. pyruvate dehydrogenase, dihydrolipoic acid dehydrogenase, dihydrothionyl dehydrogenase, acetaldehyde dehydrogenase, and acetyl CoA synthase were also downregulated (Fig. 4), indicating a decrease in the synthesis of acetyl CoA. Most of the enzymes that are involved in the fatty acid biosynthesis pathway were downregulated (see Additional file 1). Acetyl-CoA is a precursor for fatty acid biosynthesis, and reduction of the biosynthesis of acetyl-CoA may be responsible for the decrease of fatty acid biosynthesis. The down-regulation fold changes of citrate synthase and dihydrothionyl dehydrogenase in TCA cycle were 1.33 and 1.52. Malate dehydrogenase was upregulated slightly. The down-regulation of citrate synthase which control the initiation of TCA cycle suggested a weakening of the TCA cycle, which might be one of the reasons for the reduced growth rate under illumination (see Additional file 1).
The results showed that ribulose-phosphate 3-epimerase and phosphoribulokinase, which catalyze the production of ribulose bisphosphate (RuBP) in the carbon fixation pathway of photosynthetic organisms, were upregulated 1.55 and 3.56, times respectively (Fig. 5). The RuBP carboxylase and glyceraldehyde-3-phosphate dehydrogenase in the Calvin cycle were upregulated 2.78 and 2.62 times respectively, indicating that the synthesis of RuBP was increased and the Calvin cycle was also enhanced. In the Calvin cycle, CO2 is converted into G3P, which is also an intermediate product of glycolysis. Under illumination, the G3P produced by the Calvin cycle may promote the reverse reaction of fructose-bisphosphate aldolase in glycolysis, thereby inhibiting glucose metabolism. Therefore, light might inhibit the glucose metabolism via chemical products of the dark reaction of photosynthesis.
Redox analysis of A. protothecoides under light and dark conditions
Previous studies have shown that higher plants also undergo redox changes during the night and day, thereby regulating the activity of sucrose transporters . Redox states integrate and coordinate the energy production and status within a cell to regulate homeostasis and metabolism . The results obtained from the DAVID, PPI and KEGG analysis, showed that the proteins involved in the redox system had significant differences, indicating that the redox state of A. protothecoides significantly changed under illumination. Accordingly, the redox properties inside and outside the cells were examined.
The pH value and Oxidation-Reduction Potential (ORP) of the culture supernatant in HC and MC were measured using a pH meter. The ORP value reflects the relative degree of oxidative and reductive properties in the medium. A positive and higher ORP value indicates that it is an oxidative environment and has higher oxidative potential, respectively. As shown in Fig. 6, the pH of the HC gradually decreased, indicating that the acidity increased. At the same time, the ORP value gradually increased, indicating that the oxidative potential increased. While the pH of the MC also gradually decreased, its ORP value increased much less, showing that the oxidation potential of MC was not significantly increased. However, the ORP value was positive, indicating that both the HC and MC were in an oxidative environment, whereby an increase of oxidation in the medium was prevented under light illumination. The improved glucose uptake of A. protothecoides in the dark might due to the stronger oxidative properties of the medium, which increased the activity of the glucose transport system.
Next, the intracellular NAD+ and NADH levels were measured. The total amount of NAD+ and NADH did not change significantly upon illumination (Fig. 6C). However, the ratio of NADH/NAD+ and contents of NADH in the MC was higher than in the HC (Figs. 6D, E and F). These results showed that illumination affects the cellular homeostasis, which ultimately led to a more reductive intracellular environment. The high intracellular reduction state of the MC might reduce the activity of the glucose transport system. This was similar to the sucrose transporter activity in plant systems, which was lower in reductive environments [21, 36].